A Human-Curated Annotation of the Candida albicans Genome

Recent sequencing and assembly of the genome for the fungal pathogen Candida albicans used simple automated procedures for the identification of putative genes. We have reviewed the entire assembly, both by hand and with additional bioinformatic resources, to accurately map and describe 6,354 genes and to identify 246 genes whose original database entries contained sequencing errors (or possibly mutations) that affect their reading frame. Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that might be targeted for antifungal therapy. We also observed that, compared to other fungi, the protein-coding sequences in the C. albicans genome are especially rich in short sequence repeats. Finally, our improved annotation permitted a detailed analysis of several multigene families, and comparative genomic studies showed that C. albicans has a far greater catabolic range, encoding respiratory Complex 1, several novel oxidoreductases and ketone body degrading enzymes, malonyl-CoA and enoyl-CoA carriers, several novel amino acid degrading enzymes, a variety of secreted catabolic lipases and proteases, and numerous transporters to assimilate the resulting nutrients. The results of these efforts will ensure that the Candida research community has uniform and comprehensive genomic information for medical research as well as for future diagnostic and therapeutic applications

In vitro transcription of a TATA-less promoter: negative regulation by the Not1 protein

Genetic experiments in the yeast Saccharomyces cerevisiae have identified the five Not proteins as global repressors of transcription which preferentially repress core promoters which do not contain a canonical TATA sequence. Recently, the Ccr4 and Caf1 proteins, required for non-fermentative gene expression, were found to be associated with the five Not proteins in 1.2 and 2 MDa Ccr4-Not complexes. These Ccr4-Not complexes, as many other global regulators of transcription, appear to regulate transcription both positively and negatively in vivo. To further characterize the activity of Not1p, the only essential known protein of the Ccr4-Not complex, and determine whether it can act directly as a transcriptional repressor, we established an in vitro transcription system in which the HIS3 TATA-less promoter can be efficiently transcribed. We demonstrate that transcription from the HIS3 TATA-less promoter can be specifically increased in vitro by preparing nuclear extracts from a conditional mutant of the NOT1 gene and analyzing transcription after shifting the nuclear extracts to the restrictive temperature. This result is the first demonstration that one of the Not proteins directly represses transcription. Moreover, it now defines an experimental system in which TATA-less transcription initiation and repression by the Ccr4-Not complex can be studied further

Candida morphogenesis and host-pathogen interactions

NRC publication: Ye

Characterization of NOT5 that encodes a new component of the Not protein complex

The yeast HIS3 gene has two core promoters: TC, a TATA-less element and TR, a canonical TATA element. Four genes encode global negative regulators of transcription that preferentially repress TC-dependent transcription: NOT1 (CDC39), NOT2 (CDC36), NOT3 and NOT4 (SIG1, MOT2). Genetic and biochemical experiments suggest that the products of these genes are associated in a complex and regulate TFIID function. In this paper, we describe a new gene, NOT5, that also represses transcription of the HIS3 TATA-less promoter preferentially and encodes a protein whose N-terminal region is 44% identical to that of Not3p. Our results indicate that NOT5 is involved in Not function and encodes a product that is physically associated with the other Not proteins. First, overexpression of NOT3 or NOT4 suppresses mutations in NOT5. Secondly, mutations in NOT4 are synthetically lethal with mutations in NOT5. Thirdly, NOT5 interacts with NOT1 and NOT3 in the two-hybrid assay. Finally, Not1p, Not3p and Not4p co-immunoprecipitate with Not5p

Transcript Profiles of Candida albicans Cortical Actin Patch Mutants Reflect Their Cellular Defects: Contribution of the Hog1p and Mkc1p Signaling Pathways

In Candida albicans, Myo5p and Sla2p are required for the polarized localization and function of cortical actin patches, for hyphal formation, and for endocytosis. Deletion of either the MYO5 or the SLA2 gene generated a common transcriptional response that involved changes in the transcript levels of cell wall protein- and membrane protein-encoding genes. However, these profiles were distinct from those observed for a mutant with specific deletions of the actin-organizing domains of Myo5p or for wild-type cells treated with cytochalasin A, both of which also generate defects in the organization of cortical actin patches. The profiles observed for the myo5Δ and sla2Δ mutants had similarities to those of wild-type cells subjected to an osmotic shock, and the defects in cortical patch function found with myo5Δ and sla2Δ mutants, but not cortical actin patch distribution per se, affected sensitivity to various stresses, including heat and osmotic shocks and cell wall damage. Secondary effects coupled with defective endocytosis, such as lack of polarized lipid rafts and associated protein Rvs167-GFP (where GFP is green fluorescent protein) and lack of polarized wall remodeling protein GFP-Gsc1, were also observed for the myo5Δ and sla2Δ mutants. The mitogen-activated protein kinases Hog1p and Mkc1p, which mediate signaling in response to osmotic stress and cell wall damage, do not play a major role in regulating the transcript level changes in the myo5Δ and sla2Δ mutants. Hog1p was not hyperphosphorylated in the myo5Δ and sla2Δ mutants, and the transcript levels of only a subset of genes affected in the myo5Δ mutant were dependent upon the presence of Hog1p and Mkc1p. However, it appears that Hog1p and Mkc1p play important roles in the myo5Δ mutant cells because double deletion of myosin I and either Hog1p or Mkc1p resulted in very-slow-growing cells

Tools for surveying and improving the quality of life: people with special needs in focus

Purpose – This article seeks to describe online tools for surveying and improving quality of life for people with disabilities living in assisted living centers and special education service organizations. Design/methodology/approach – Ensuring a decent quality of life for disabled people is an important welfare state goal. Using well-accepted quality of life conceptions, online diagnostic and planning tools were developed during an Institute for Education, University of Zurich, research project. Findings – The diagnostic tools measure, evaluate and analyze disabled people's quality of life. The planning tools identify factors that can affect their quality of life and suggest improvements. Research limitations/implications – Instrument validity and reliability are not tested according to the standard statistical procedures. This will be done at a more advanced stage of the project. Instead, the tool is developed, refined and adjusted in cooperation with practitioners who are constantly judging it according to best practice standards. Practical implications – The tools support staff in assisted living centers and special education service organizations. Originality/value – These tools offer comprehensive resources for surveying, quantifying, evaluating, describing and simulating quality of life elements

Functional Characterization of Myosin I Tail Regions in Candida albicans

The molecular motor myosin I is required for hyphal growth in the pathogenic yeast Candida albicans. Specific myosin I functions were investigated by a deletion analysis of five neck and tail regions. Hyphal formation requires both the TH1 region and the IQ motifs. The TH2 region is important for optimal hyphal growth. All of the regions, except for the SH3 and acidic (A) regions that were examined individually, were required for the localization of myosin I at the hyphal tip. Similarly, all of the domains were required for the association of myosin I with pelletable actin-bound complexes. Moreover, the hyphal tip localization of cortical actin patches, identified by both rhodamine-phalloidin staining and Arp3-green fluorescent protein signals, was dependent on myosin I. Double deletion of the A and SH3 domains depolarized the distribution of the cortical actin patches without affecting the ability of the mutant to form hyphae, suggesting that myosin I has distinct functions in these processes. Among the six myosin I tail domain mutants, the ability to form hyphae was strictly correlated with endocytosis. We propose that the uptake of cell wall remodeling enzymes and excess plasma membrane is critical for hyphal formation

Comparative ultrastructure of fibrin networks of a dog after thrombotic ischaemic stroke

A cerebrovascular accident or stroke is a rare condition in dogs, but previous studies suggest that it is now increasingly being recognised. Platelets and fibrin networks are involved in haemostasis, which is disrupted during a thrombotic event. In this study we investigate the ultrastructure of the fibrin networks of a dog that had suffered ischaemic stroke, following suspected thromboembolism from clots that became dislodged during catheter maintenance (flushing with heparinised saline) 2 days after carotid artery catheter insertion. Fibrin networks of blood samples that were collected immediately after the stroke, 15 min after treatment with streptokinase and 24 h after treatment, were studied. The results were compared to those of two control dogs. During a stroke, fibrin morphology changes to form a thick, matted layer. Post-treatment ultrastructure shows that the fibrin morphology returns to that comparable to controls. Our results show that during thrombotic risk, fibrin network morphology changes visibly and reduces the fibrinolytic activity of the coagulation system